Park Hongmarn, McGibbon Louise C, Potts Anastasia H, Yakhnin Helen, Romeo Tony, Babitzke Paul
Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida, USA.
mBio. 2017 Aug 29;8(4):e01355-17. doi: 10.1128/mBio.01355-17.
CsrA is a global regulatory RNA binding protein that has important roles in regulating carbon metabolism, motility, biofilm formation, and numerous other cellular processes. IraD functions as an antiadapter protein that inhibits RssB-mediated degradation of RpoS, the general stress response and stationary-phase sigma factor of Here we identified a novel mechanism in which CsrA represses translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and fusions demonstrated that CsrA represses expression. Gel mobility shift, footprint, and toeprint studies identified four CsrA binding sites in the leader transcript, all of which are far upstream of the ribosome binding site. Computational modeling and RNA structure mapping identified an RNA structure that sequesters the Shine-Dalgarno (SD) sequence. Three open reading frames (ORFs), all of which are translated, were identified in the leader region. Two of these ORFs do not affect expression. However, the translation initiation region of the third ORF contains three of the CsrA binding sites, one of which overlaps its SD sequence. Furthermore, the ORF stop codon overlaps the start codon, a sequence arrangement indicative of translational coupling. expression and translation studies with wild-type and mutant reporter fusions demonstrated that bound CsrA directly represses translation initiation of this ORF. We further established that CsrA-dependent repression of translation occurs entirely via translational coupling with this ORF, leading to accelerated mRNA decay. CsrA posttranscriptionally represses gene expression associated with stationary-phase bacterial growth, often in opposition to the transcriptional effects of the stationary-phase sigma factor RpoS. We show that CsrA employs a novel regulatory mechanism to repress translation of , which encodes an antiadapter protein that protects RpoS against proteolysis. CsrA binds to four sites in the leader transcript but does not directly occlude ribosome binding to the SD sequence. Instead, CsrA represses translation of a short open reading frame encoded upstream of , causing repression of translation via translational coupling. This finding offers a novel mechanism of gene regulation by the global regulator CsrA, and since RpoS can activate transcription, this also highlights a new negative-feedback loop within the complex Csr and RpoS circuitry.
CsrA是一种全局性调控RNA结合蛋白，在调节碳代谢、运动性、生物膜形成及许多其他细胞过程中发挥重要作用。IraD作为一种抗衔接蛋白，可抑制RssB介导的RpoS降解，RpoS是一般应激反应及稳定期的σ因子。在此，我们鉴定出一种新机制，即CsrA通过翻译偶联抑制翻译。利用定量逆转录PCR、蛋白质免疫印迹及融合蛋白进行的表达研究表明，CsrA抑制表达。凝胶迁移率变动分析、足迹分析及脚印分析确定了前导转录本中的四个CsrA结合位点，所有这些位点均位于核糖体结合位点的上游很远位置。计算建模和RNA结构图谱分析确定了一种封存Shine-Dalgarno（SD）序列的RNA结构。在前导区域鉴定出三个均被翻译的开放阅读框（ORF）。其中两个ORF不影响表达。然而，第三个ORF的翻译起始区域包含三个CsrA结合位点，其中一个与它的SD序列重叠。此外，ORF终止密码子与起始密码子重叠，这种序列排列表明存在翻译偶联。利用野生型和突变型报告基因融合蛋白进行的表达和翻译研究表明，结合的CsrA直接抑制该ORF的翻译起始。我们进一步确定，CsrA依赖的翻译抑制完全通过与该ORF的翻译偶联发生，导致mRNA加速衰变。CsrA在转录后抑制与细菌稳定期生长相关的基因表达，这通常与稳定期σ因子RpoS的转录作用相反。我们表明，CsrA采用一种新调控机制抑制的翻译，该基因编码一种抗衔接蛋白，可保护RpoS不被蛋白酶解。CsrA结合在前导转录本的四个位点，但不直接阻止核糖体与SD序列结合。相反，CsrA抑制上游编码的一个短开放阅读框的翻译，通过翻译偶联导致的翻译受到抑制。这一发现揭示了全局调控因子CsrA调控基因的新机制，并且由于RpoS可激活转录，这也凸显了复杂的Csr和RpoS调控回路中的一个新的负反馈环。
